Background: Stroke therapies remain an area of ongoing research. Gene therapies offer a novel approach to repair tissue damage, particularly NeuroD1-mediated astrocyte-to-neuron conversion, which regenerates functional neurons after ischemic injury. Here, we applied NeuroD1 therapy in a non-human primates (NHPs) stroke model to evaluate its effects on corticospinal tract (CST) recovery and motor performance. Methods: Eight NHPs underwent middle cerebral artery occlusion (MCAO). Fourteen days later, six animals received intracranial NeuroD1 treatment (three high-dose, three low-dose), while two received a control solution. Neurological and functional performance were assessed daily. MRI scans were performed at baseline and at 7, 30, 90, 120, and 240 days post-MCAO, with the bilateral CST reconstructed at each time point. All procedures followed Canadian Council of Animal Care guidelines and were approved by Queen’s University’s Animal Use Subcommittee. Results: We found that NHPs receiving the control solution exhibited poorer motor recovery and minimal CST reconstruction. In contrast, those treated with a low dose of NeuroD1 demonstrated motor and functional recovery along with CST reconstruction. Notably, animals receiving the higher dose showed the most significant overall recovery including a greater CST integrity. Conclusions: NeuroD1 treatment promotes white matter tract restoration and facilitates motor recovery following stroke.